GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis

Abstract

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-offunction mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3 T180A had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.signal transduction | phosphorylation cascades | kinase activation

BRASSINOSTEROID INSENSITIVE2 Interacts with ABSCISIC ACID INSENSITIVE5 to Mediate the Antagonism of Brassinosteroids to Abscisic Acid during Seed Germination inArabidopsis

“…Error bars show SD from three independent RNA extractions. result (Supplemental Figure 5), Cai et al (2014) found that BIN2 positively regulated the induced expression levels of several ABA-responsive genes in germinated seedlings (10-d-old seedlings). Interestingly, our yeast-two hybrid assays showed that BIN2 and its homologs also interact with ABSCISIC ACID RESPONSIVE ELEMENT BINDING FACTOR1 (ABF1) and/or ABF3 (Supplemental Figure 6), two critical regulators of ABA signaling in germinated seedlings.…”

“…Recently, Cai et al (2014) demonstrated that BIN2 physically interacts with subgroup III SnRK2 kinases and positively regulates ABA signaling to inhibit root elongation. Biochemical analysis in their study revealed that BIN2 phosphorylates SnRK2.2 and SnRK2.3.…”

“…Moreover, BIN2 phosphorylation was demonstrated to positively affect the activity of its interacting kinase. Cai et al (2014) recently showed that BIN2 phosphorylation enhances the kinase activity of SnRK2.3 to positively modulate ABA signaling. In addition, Cho et al (2014) found that BIN2 phosphorylation of ARF7 and ARF19 attenuates their interaction with AUXIN/INDOLE-3-ACETIC ACID repressor proteins, subsequently activating these ARF proteins and potentiating auxin signaling output to regulate lateral root development.…”

Two Distinct Families of Protein Kinases Are Required for Plant Growth under High External Mg2+ Concentrations in Arabidopsis

“…Previous reports showed that the phosphorylation of the activation loop plays a crucial role in the activation of subclass III SnRK2s (Belin et al, 2006;Umezawa et al, 2009) and that a Glycogen synthase kinase3-like kinase, brassinosteroid insensitive2 (BIN2), phosphorylates the activation loop in the KD of SRK2D and activates its autophosphorylation activity and the kinase activity toward ABF2/AREB1 (Cai et al, 2014). Thus, an alternative possibility is that the phosphorylation of SRK2D by CIPK26 has functions other than activation.…”

Feedback Regulation of ABA Signaling and Biosynthesis by a bZIP Transcription Factor Targets Drought-Resistance-Related Genes

“…For example, BRASSINOSTEROID INSENSITIVE2 was found to positively modulate ABA signaling through phosphorylation of subgroup III SnRKs in Arabidopsis (Cai et al, 2014), and ARK plays an essential role in the activation of SnRK2 in the moss Physcomitrella patens (Cai et al, 2014;Saruhashi et al, 2015). Recently, ABI1 was found to be degraded through ubiquitination by PUB12 and PUB13 in the presence of ABA and an ABA receptor in Arabidopsis, which may be regarded, to our knowledge, as a new positive regulation pathway of ABA signaling (Kong et al, 2015).…”